1. Field of the Invention
The present invention relates to methods and materials useful for performing reverse transcription-polymerase chain reaction in prokaryotic cells.
2. Description of the Related Art
The first comprehensive studies of cellular response resulted from the development of two-dimensional gel electrophoresis and a complementary method for quantitatively measuring protein levels (O""Farell, P. H., xe2x80x9cHigh Resolution Two-Dimensional Electrophoresis of Proteinsxe2x80x9d, Journal of Biological Chemistry, 250(10):4007-21 (1975); and Pederson el al., in 1978). In parallel, Casadaban created transcriptional fusion proteins to assist in the study of gene regulation, which has been particularly useful for analyzing genes whose products are difficult to characterize (Casadaban, M. J., xe2x80x9cRegulation of the Regulatory Gene for the Arabinose Pathway, araCxe2x80x9d, Journal of Molecular Biology, 104:557-66 (1976)). Both of these techniques have since been further refined (See Casadaban, M. J. et al., xe2x80x9cLactose Genes Fused to Exogenous Promoters in One Step Using a Mu-lac Bacteriophage: In vivo Probe for Transcriptional Control Sequencesxe2x80x9d, Proceedings of the National Academy of Sciences, 76(9):4530-33 (1979); and see Kenyon, C. J. et al., xe2x80x9cDNA-damaging Agents Stimulate Gene Expression at Specific Loci in Escherichia colixe2x80x9d, Proceedings of the National Academy of Sciences, 177(5):2819-23 (1980)).
More recently, detection of transcriptional regulation has been further simplified due to such bioluminescent reporter proteins as luciferase (Rupani, S. et al, xe2x80x9cCharacterization of the Stress Response of a Bioluminescent Biological Sensor in Batch and Continuous Culturesxe2x80x9d, Biotechnology Progress, 12:387-92 (1996); and VanDyk, T. K. et al.,xe2x80x9cRapid and Sensitive Pollutant Detection by Induction of Heat Shock Gene-bioluminescence Gene Fusionsxe2x80x9d, Applied Environmental Microbiology, 60:1414-20 (1994)) and green fluorescent protein (Gill, R. T. et al., xe2x80x9cPhysiological Effects of DTT Addition to E. coli Including Growth Rate, Specific Oxygen Uptake, Heat Shock Protein Expression, and Specific Activity of Recombinant Proteinxe2x80x9d, Biotechnology and Bioengineering, 59:248-59 (1998)).
In the mid-1980""s, Kohara et al. developed a restriction map of 3400 xcex bacteriophage clones containing segments of the E. coli chromosome (Kohara et al., xe2x80x9cThe Physical Map of the Whole E. coli Chromosome: Application of a New Strategy for Rapid Analysis and Sorting of a Large Genomic Libraryxe2x80x9d, Cell, 50:495-508 (1987)). This set was originally developed to not only map the location of E. coli genes but also to map and clone the gene or genes that is (are) induced in response to a certain external or internal signal(s). According to Kohara et al., their identified clones could be exploited for the isolation of any desired E. coli genes if their map positions were known.
Using the Kohara set of overlapping xcex phage clones, Chuang el al. later demonstrated that global gene regulation in E. coli could be analyzed using single stranded reverse transcribed complementary DNA (hereinafter cDNA), in which radiolabeled cDNA was hybridized with the Kohara clones (Chuang, S. el al., xe2x80x9cGlobal Regulation of Gene Expression in Escherichia colixe2x80x9d, Journal of Bacteriology, 175:2026-36 (1993)). These clones, containing the entire E. coli genome, were used to map the location of cDNA homologs. Through follow-up Southern blotting (Southern, E. M., xe2x80x9cDetection of Specific Sequences Among DNA Fragments Separated by Gel Electrophoresisxe2x80x9d, Journal of Molecular Biology, 98:503-17, (1975)), Chuang et al. identified 26 new heat shock genes for E. coli (Chuang, S. et al., xe2x80x9cGlobal Regulation of Gene Expression in Escherichia colixe2x80x9d, Journal of Bacteriology, 175:2026-36, (1993)). While this technique was a significant improvement over the previous methodologies of two-dimensional electrophoresis or transcriptional fusions for analyzing global genetic regulation, the messenger RNA (hereinafter mRNA) signal to total RNA noise ratio remained small.
One year later, Wong et al. applied a random arbitrary primed PCR amplification step, after reverse transcription of total RNA to detect a stress induced gene in Salmonella typhimurium (Wong, K. K. et al., xe2x80x9cStress-inducible Gene of Salmonella typhimurium Identified by Arbitrarily Primed PCR of RNAxe2x80x9d, Proceedings of the National Academy of Science U.S.A., 91:639-43 (1994). While this technique did improve the level of mRNA signal, the signal to noise ratio did not change due to xe2x80x9crandomxe2x80x9d amplification of RNA templates. In addition, due to the use of sequencing gels for transcript identification, this technique did not permit quantification at the genomic level.
An innovative refinement of these two methods was recently reported by de Saizieu et al., in which non-radioactively labeled total prokaryotic RNA was hybridized directly to an oligonucleotide array synthesized and bonded to a silicon chip (de Saizieu, A. et al., xe2x80x9cBacterial Transcript Imaging by Hybridization of Total RNA to Oligonucleotide Arrays, Nature Biotechnology, 16(l):45-8 (1998). This analysis, which allowed quantification for a large subset of transcribed genes, additionally required scanning confocal microscopy as RNA levels were detected as unamplified transcripts.
In the meantime, differential display techniques based on polymerase chain reaction (hereinafter PCR) amplification have advanced rapidly in eukaryotic systems due to mRNA polyadenylation that exclusively occurs in eukaryotic organisms. Reverse transcription-polymerase chain reaction (hereinafter RTPCR) (Liang, P. et al., xe2x80x9cDifferential Display of Eukaryotic Messenger RNA by Means of the Polymerase Chain Reactionxe2x80x9d, Science, 257:967-71 (1992)) and random arbitrary-primed polymerase chain reaction (hereinafter RAP-PCR) (Welsh, J. et al., xe2x80x9cArbitrarily Primed PCR Fingerprinting of RNA, Nucleic Acids Researchxe2x80x9d, 20:4965-70 (1992)) specifically were developed in response to the problem of obtaining a small mRNA signal ratio to total RNA noise ratio. These techniques enhanced the mRNA signal to total RNA noise ratio via differential display experiments. With these techniques, the identification of differentially expressed genes among the mRNA for a pair of eukaryotes was carried out, with subsequent recovery of the cDNA and genomic clones for each eukaryote (Liang et al. 1992).
Additional developments in eukaryotic-based RTPCR/RAP-PCR included the use of random arbitrary primers or xe2x80x9cmotifxe2x80x9d primers, the use of sense and antisense oligonucleotide primers, usually degenerate in sequence, which were designed to amplify cDNA templates encoding proteins using particular structural motifs, and amplification products were displayed using agarose gel electrophoresis and ethidium bromide fluorescence staining (Donohue, P. et aL, Differential Display Methods and Protocols, Chapter 3: xe2x80x9cDifferential Display Using Random Hexamer-Primed cDNA, Motif Primers, and Agarose Gel Electrophoresis, 85: 25-35, 25-6 (Peng Liang and Arthur B. Pardee ed., 1997).
Pardee et al, U.S. Pat. No. 5,262,311, disclose a method of isolating mRNAs as cDNAs by employing a polymerase amplification method using two specific primers. This method is used in eukaryotic organisms and is known as differential display. Differential display involves amplifying partial cDNA sequences from subsets of mRNAs by reverse transcription and the polymerase reaction, then displaying these sequences on a sequencing gel.
McClelland et al., U.S. Pat. No. 5,487,985, disclose an arbitrarily primed polymerase chain reaction (hereinafter AP-PCR) for a method of generating a set of discrete DNA amplification products characteristic of a genome as a xe2x80x9cfingerprintxe2x80x9d. This method is suitable for the identification of bacterial species, bacterial strains, mammals, and plants and utilizes a single-stranded DNA primer.
Villeponteau et al., U.S. Pat. No. 5,580,726, disclose a method and kit for enhanced differential display for eukaryotic organisms which comprises using first oligonucleotide primers for reverse transcription of mRNAs and both the first and second oligonucleotide primers for amplification of the resultant cDNAs. It is designed as a technique for screening differences in gene expression between different stages of cell development.
Liang et aL, U.S. Pat. No. 5,599,672, disclose a method of isolating mRNAs as cDNAs, by using two oligodeoxynucleotide primers of varying combinations wherein the first primer is used as a primer for reverse transcription of the mRNA and the resultant cDNA is amplified with a polymerase using both the first and second primers as a primer set.
Pardee et al, U.S. Pat. No. 5,665,547, disclose a method for comparing amounts or levels of mRNAs in eukaryotic organisms that employs a polymerase amplification method using two oligodeoxynucleotide primers of varying combinations in which the first primer is used as a primer for reverse transcription and the resultant cDNA is amplified using both the first and second primers as a primer set.
Belyavasky et al., U.S. Pat. No. 5,814,445 disclose a method of identification of differentially expressed mRNA in eukaryotic organisms by arbitrary primed RT-PCR. This consists of synthesizing, from a set of sequences of mRNA, sets of fragments of cDNA which are separated with the aid of gel electrophoresis and a detection of a marker and comparison of the separation pictures is carried out. If further detection is necessary, the separated cDNA fragments are transferred to a membrane with sequential hybridization by oligonucleotides which partially overlap the common sequence of the fragments. Additional analysis of the separation picture is available.
McClelland et al., U.S. Pat. No. 5,861,245, disclose AP-PCR as a method of generating a set of discrete DNA amplification products characteristic of a genome as a xe2x80x9cfingerprintxe2x80x9d. This method is suitable for the identification of bacterial species, bacterial strains, mammals and plants and utilizes a single-stranded DNA primer.
It is an object of the present invention to provide a method for the rapid identification of genes with increased or decreased transcription in response to environmental stimuli.
It is another object of the present invention to provide a method that may be utilized in bioprocess fermentations as well as in basic science studies of prokaryotic global genetic regulation.
It is yet another object of the present invention to provide a kit for analysis of genetic regulation and/or the response of genes to specific stimuli.
According to a first broad aspect of the present invention, there is provided a method comprising the steps of: adding a first primer mixture comprising RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, RT10, PCR1, PCR3 and PCR5 to a first nucleic acid sample including a first mixture of mRNA to form a first primer/first nucleic acid sample mixture; adding the first primer mixture to a second nucleic acid sample including a first mixture of mRNA to form a first primer/second nucleic acid sample mixture; incubating the first primer/first nucleic acid sample mixture to produce a first population of cDNA; incubating the first primer/second nucleic acid sample mixture to produce a second population of cDNA; adding a second primer mixture comprising RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, RT10, PCR1, PCR2, PCR3, PCR4, PCR5, PCR6, PCR7, PCR8, PCR9, and PCR10 to the first population of cDNA to form a second primer/first population of cDNA mixture; adding the second primer mixture to the second population of cDNA to form a second primer/second population of cDNA mixture; amplifying the second primer/first population of cDNA mixture to produce a third population of cDNA; amplifying the second primer/second population of cDNA mixture to produce a fourth population of cDNA; identifying the presence or level of mRNA in the third population of cDNA; and identifying the presence or level of mRNA in the fourth population of cDNA.
According to a second broad aspect of the present invention, there is provided a composition comprising primers RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, RT10, PCR1, PCR3, and PCR5.
According to a third broad aspect of the present invention, there is provided a composition comprising primers RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, RT10, PCR1, PCR2, PCR3, PCR4, PCR5, PCR6, PCR7, PCR8, PCR9, and PCR10.
According to a fourth broad aspect of the present invention, there is provided a kit for enabling RTPCR of prokaryotic mRNA, comprising: a first container containing a first primer mixture comprising: RT1, RT2, RT3, RT4, RT5, RT6, RT7, RF8, RT9, RT10, PCR1, PCR3 and PCR5; and a second container containing a second primer mixture comprising: RT1, RT2, RT3, RT4, RT5, RT6, RT7, RT8, RT9, RT10, PCR1, PCR2, PCR3, PCR4, PCR5, PCR6, PCR7, PCR8, PCR9, and PCR10.
According to a fifth broad aspect of the present invention, there is provided a method comprising the steps of: adding a first primer mixture to a first nucleic acid sample including a first mixture of mRNA to form a first primer/first nucleic acid sample mixture; adding the first primer mixture to a second nucleic acid sample including a first mixture of mRNA to form a first primer/second nucleic acid sample mixture; incubating the first primer/first nucleic acid sample mixture to produce a first population of cDNA; incubating the first primer/second nucleic acid sample mixture to produce a second population of cDNA; adding a second primer mixture to the first population of cDNA to form a second primer/first population of cDNA mixture; adding the second primer mixture to the second population of cDNA to form a second primer/second population of cDNA mixture; amplifying the second primer/first population of cDNA mixture to produce a third population of cDNA; amplifying the second primer/second population of cDNA mixture to produce a fourth population of cDNA; identifying the presence or level of mRNA in the third population of cDNA using a gene plotting membrane; and identifying the presence or level of mRNA in the fourth population of cDNA using a gene plotting membrane.
Other objects and features of the present invention will be apparent from the following detailed description of the preferred embodiment.